Our long term objective is to understand the role of the renal epithelial sodium channel (ENaC) and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl channel in regulating Na and Cl homeostasis in blood pressure in normal and disease states. ENaC and the CFTR Cl channel are expressed in the apical membrane of principal cells in the renal cortical collecting duct. Most ion channels, including ENaC, are multimeric complexes containing one or more pore-forming subunits and a variable number of regulatory subunits. The regulatory subunits play a variety of roles including facilitating subunit oligomerization and exit from the endoplasmic reticulum (ER). Recent studies have identified an ER retention motif (RXR) that is hidden or overcome by anterograde trafficking signals in correctly assembled ion channel complexes. This mechanisn allows only correctly oligomerized channel complexes to traffic to the plasma membrane. The RXR motif is highly conserved in all three ENaC subunits. However, the role of the RXR motif and the mechanisms that regulate ENaC subunit assembly and ER export are poorly understood. Accordingly, the first goal of this application is to test the hypothesis that ER retention motifs (RXR) in ENaC regulate export fron the endoplasmic reticulum, biosynthesis and degradation. During the last funding period, we demonstrated that ENaC enhances CFTR Cl currents 6-fold in part by increasing the number of CFTR channels in the plasma membrane. However, the molecular and cellular mechanisms involved in the functional interaction between ENaC and CFTR are incompletely understood. Therefore, the second goal of this application is to test the hypothesis that ENaC enhances the number of CFTR channels in the plasma membrane by modulating the intracellular trafficking of CFTR. To these ends we will use two cell model systems, including Xenopus oocytes and MDCK cells, and utilize a combination of cellular, molecular, biochemical and electrophysiological approaches. The experiments described in this proposal will provide new information on the cellular and molecular mechanisms responsible for Na and Cl transport by the renal cortical collecting duct. Moreover, we anticipate that these studies will elucidate the role of ER retention motifs as a mechanism to regulate the membrane expression of correctly oligomerized ENaC channels.